Devices and methods for dual excitation Raman spectroscopy

ABSTRACT

Spectroscopic analysis systems and methods for analyzing samples are disclosed. An analysis system may contain an electromagnetic radiation source to provide radiation, a spectroscopic analysis chamber to perform a coherent Raman spectroscopy (e.g., stimulated Raman or coherent anti-Stokes Raman spectroscopy), and a radiation detector to detect radiation based on the spectroscopy. The chamber may have a resonant cavity to contain a sample for analysis, at least one window to the cavity to transmit the first radiation into the cavity and to transmit a second radiation out, a plurality of reflectors affixed to a housing of the cavity to reflect radiation of a predetermined frequency, the plurality of reflectors separated by a distance that is sufficient to resonate the radiation. The spectroscopic analysis system may be coupled with a nucleic acid sequencing system to receive a single nucleic acid derivative in solution and identify the derivative to sequence the nucleic acid.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 11/404,680, filed Apr. 14, 2006 now U.S. Pat. No. 8,009,288 which is a divisional application of U.S. application Ser. No. 10/675,884, filed Sep. 29, 2003 now abandoned which is a continuation-in-part application of U.S. application Ser. No. 10/262,349 filed Sep. 30, 2002 now U.S. Pat. No. 8,088,628. The disclosure of each of the prior applications is considered part of and is incorporated by reference in the disclosure of this application.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Embodiments of the invention relate to analyzing a sample with Raman spectroscopy. In particular, embodiments relate to analyzing a sample containing a single nucleic acid derivative of interest contained within a spectroscopic analysis chamber with stimulated or coherent anti-Stokes Raman spectroscopy.

2. Background Information

Deoxyribonucleic acid (DNA) sequencing has many commercially important applications in the fields of medical diagnosis, drug discovery, and treatment of disease. Existing methods for DNA sequencing, based on detection of fluorescence labeled DNA molecules that have been separated by size, are limited by the length of the DNA that can be sequenced. Typically, only 500 to 1,000 bases of DNA sequence can be determined at one time. This is much shorter than the length of the functional unit of DNA, referred to as a gene, which can be tens or even hundreds of thousands of bases in length. Using current methods, determination of a complete gene sequence requires that many copies of the gene be produced, cut into overlapping fragments and sequenced, after which the overlapping DNA sequences may be assembled into the complete gene. This process is laborious, expensive, inefficient and time-consuming. It also typically requires the use of fluorescent or radioactive labels, which can potentially pose safety and waste disposal problems.

More recently, methods for DNA sequencing have been developed involving hybridization to short oligonucleotides of defined sequence, attached to specific locations on DNA chips. Such methods may be used to infer short DNA sequences or to detect the presence of a specific DNA molecule in a sample, but are not suited for identifying long DNA sequences.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may best be understood by referring to the following description and accompanying drawings that are used to illustrate embodiments of the invention. In the drawings:

FIG. 1 shows a method for identifying a sample based on a resonance enhanced spectroscopic analysis, according to embodiments of the invention.

FIG. 2 shows a cross-sectional view of a spectroscopic analysis system containing a chamber in a fluid channel, according to embodiments of the invention.

FIG. 3 shows Rayleigh, Stokes, and anti-Stokes radiation.

FIG. 4 shows Stokes Raman spectra for dilute aqueous solutions of four DNA nucleotides, according to embodiments of the invention.

FIG. 5 shows a cross-sectional view of a spectroscopic analysis system containing a spheroidal chamber coupled with a fluid channel, according to embodiments of the invention.

FIG. 6 shows another cross-sectional view of a spheroidal chamber coupled with a fluid channel, according to embodiments of the invention.

FIG. 7A shows a view of the top of the chamber shown in FIG. 6 along the section lines 7 A-7 A, according to embodiments of the invention.

FIG. 7B shows a view of the bottom of the chamber shown in FIG. 6 along the section lines 7B-7B, according to embodiments of the invention.

FIG. 8 shows a top-plan view of a spectroscopic analysis chamber configured to receive excitation radiation in the form of aligned frequency-matched seed radiation having the same frequency as the scattered radiation, and transverse frequency-unmatched radiation having a different frequency than the scattered radiation, according to embodiments of the invention.

FIG. 9 shows an energy level diagram for a coherent anti-Stokes Raman Spectroscopy process used to analyze a sample, according to embodiments of the invention.

FIG. 10 shows a cross-beam configuration for implementing coherent anti-Stokes Raman spectroscopy, according to embodiments of the invention.

FIG. 11 shows momentum conservation for a cross-beam configuration similar to that shown in FIG. 10, according to embodiments of the invention.

FIG. 12 shows a forward-scattered configuration for implementing coherent anti-Stokes Raman spectroscopy, according to embodiments of the invention.

FIG. 13 shows a back-scattered configuration for implementing coherent anti-Stokes Raman spectroscopy, according to embodiments of the invention.

FIG. 14 shows a nucleic acid sequencing system in which embodiments of the invention may be implemented.

FIG. 15 shows an exemplary apparatus and method for nucleic acid sequencing by surface enhanced Raman spectroscopy (SERS), surface enhanced resonance Raman spectroscopy (SERRS), resonance enhanced Raman spectroscopy (e.g., stimulated Raman spectroscopy), and/or coherent anti-Stokes Raman spectroscopy (CARS) detection.

FIG. 16 shows the Raman spectra of all four deoxynucleoside monophosphates (dNTPs) at 100 mM concentration, using a 100 millisecond data collection time.

FIG. 17 shows SERS detection of 1 nM guanine, obtained from dGMP by acid treatment according to Nucleic Acid Chemistry, Part 1, L. B. Townsend and R. S. Tipson (Eds.), Wiley-Interscience, New York, 1978.

FIG. 18 shows SERS detection of 100 nM cytosine.

FIG. 19 shows SERS detection of 100 nM thymine.

FIG. 20 shows SERS detection of 100 pM adenine, obtained from dAMP by acid treatment.

FIG. 21 shows a comparative SERS spectrum of a 500 nM solution of deoxyadenosine triphosphate covalently labeled with fluorescein (upper trace) and unlabeled dATP (lower trace).

FIG. 22 shows a plot comparing the energies of Raman scattered light generated from single molecules respectively contained inside an optical resonance chamber (curves A and B) or positioned in free space without resonance enhancement (curve C), according to one embodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Described herein are spectroscopic analysis systems and methods for analyzing samples with Raman spectroscopy. In the following description, numerous specific details are set forth. However, it is understood that embodiments of the invention may be practiced without these specific details. For example, a practitioner may use a spectroscopic analysis chamber other than the specific chambers disclosed herein. As another example, a practitioner may use another spectroscopy technique than the specific stimulated and coherent anti-Stokes Raman spectroscopies disclosed herein. In other instances, well-known structures and techniques have not been shown in detail in order to avoid obscuring the understanding of this description. It is recognized that there is a generally high level of skill in the arts of spectroscopy and in fabricating chambers as disclosed herein.

Embodiments of the invention may be used in conjunction with DNA sequencing. One exemplary method for DNA sequencing may involve the ordered deconstruction of a DNA molecule into smaller nucleotide components, for example through the use of enzymes, and then analyzing and identifying the nucleotides to determine the ordered sequence of the nucleotides in the original DNA molecule. Spontaneous Raman spectroscopy has been attempted for identification of nucleotides. During spontaneous Raman spectroscopy, the sample containing the nucleotide is exposed to radiation, and a small portion of the radiation that interacts with the nucleotide is scattered due to the spontaneous Raman scattering effect according to vibrational characteristics of the nucleotide.

Unfortunately, in spontaneous Raman spectroscopy, the optical signals scattered from nucleotides in dilute solution are generally very weak, and the resulting generally low probability of detecting the optical signals makes the approach insensitive, inaccurate, and unreliable. There have been efforts to improve the accuracy of this approach by making multiple measurements and performing data averaging, although this takes more time, and may not be desirable in a rapid DNA sequencing environment. There have also been efforts to improve the strength of the optical signal by attaching chemical moieties, such as fluorescent chromophores, to the nucleotides in order to enhance their luminescent or fluorescent properties. Reliably and consistently attaching these moieties remains problematic. The moieties or their buffer solutions may be unstable. In some instances, the moieties fail to attach altogether. In other instances the moieties that do attach have different characteristics and cause their nucleotides to have a different spectroscopic response. Such problems make it difficult to accurately and reliably identify the nucleotides. In addition, the processes to attach the chemical moieties add additional time and complexity to the analysis. These problems may be overcome by the methods and systems disclosed herein.

FIG. 1 shows a method for identifying a sample based on a resonance enhanced stimulated Raman spectroscopic analysis, according to embodiments of the invention. The method allows identifying a sample based on spectroscopic data that serves as a fingerprint or signature for the sample. In brief, the method includes adding a sample, such as a single molecule of interest in solution, to a resonant spectroscopic analysis chamber at block 110, analyzing the sample with a resonance enhanced stimulated Raman spectroscopy at blocks 120-160, and identifying the sample based on the analysis at block 170. The analysis of the single molecule is an aspect of some applications, and not a limitation. Other applications may involve analyzing a plurality if molecules. In some embodiments of the invention, the resonance enhanced stimulated Raman spectroscopy may include irradiating a sample contained in a resonance chamber at block 120, scattering radiation from the sample at block 130, resonating the scattered radiation in the chamber at block 140, irradiating or transmitting the scattered radiation from the chamber at block 150, and detecting the irradiated scattered radiation at block 160. In one aspect, the method may be used in coordination with nucleic acid sequencing and may include identifying a single nucleic acid derivative in a sample received from a nucleic acid sequencing system in an effort to sequence a DNA or RNA molecule.

FIG. 2 shows a cross-sectional view of a spectroscopic analysis system 200 suitable to perform a resonance enhanced stimulated Raman spectroscopic analysis of a sample in order to identify the sample, according to embodiments of the invention. The system includes an electromagnetic radiation source 210, a fluid channel 270, a spectroscopic analysis chamber 250 (shown by dashed lines), and an electromagnetic radiation detector 290. A sample 230 is contained within the chamber. During resonance enhanced spectroscopic analysis of the sample, the electromagnetic radiation source provides input or excitation radiation 215 to irradiate the sample within the chamber in order to generate an output of Raman inelastically scattered radiation 285 that contains information that may be used to characterize and identify the sample. In the case of stimulated Raman spectroscopy, the input radiation may be visible, ultraviolet, or near infrared spectrum radiation from an electromagnetic radiation source such as a laser and the output radiation may be corresponding stimulated Raman inelastically scattered radiation. The output radiation may be detected by the detector and used to identify the sample or one or more components thereof.

Initially, with reference to block 110 of the method 100, the sample 230, which may include a liquid, gas, or mixed fluid, is added to the chamber 250. In the system illustrated in FIG. 2, the sample is added to the chamber by flowing through the fluid channel. The channel is a void or hollowed-out space within a solid material and may be, for example, any tube, pipe, duct, or conduit to convey a fluid. The cross section of the channel may have a circular, oval, square, rectangular, or other shape. The fluid channel has a sample inlet 275 that is coupled with a sample source to receive a sample for analysis and a sample outlet 280 that is coupled with a sample destination to discharge an analyzed sample. The sample may be received at the inlet, flowed through the channel, flowed into the chamber, analyzed, subsequently flowed out of the chamber, and flowed through the outlet to the proper destination. Flow may be achieved by providing an appropriate driving force, such as a pressure differential between the inlet and the outlet or a pump within the channel. As one example, an aqueous solution of diluted nucleic acid derivative may be received from a pressurized nucleic acid sequencing system by pressure injection and added to the chamber by flowing through the channel in order to identify the particular nucleic acid derivative. Alternatively, the sample may be added with a fluid pump, such as a syringe, through an opening to the chamber, rather than by flowing through a fluid channel, and subsequently removed with the syringe, hi still other implementations, if the sample is electrically charged it may be added to the chamber under the force provided by an electric field, or if the sample is magnetic (e.g., has a magnetic moiety attached) it may be added to the chamber by the force provided by a magnetic field.

Although not required, the channel may be micro-sized. The term micro-sized channel, microfluidic channel, and the like will be used to refer to a channel having a cross-sectional length, for example a diameter in the case of a tubular pipe, which is less than approximately one millimeter (m, one-thousandth of a meter). Often, the micro-sized channel may have a cross-sectional length that is in the range of approximately 10-500 micrometers (um, one millionth of a meter). To help put these lengths in proper perspective, the cross-sectional diameter of a human hair is approximately 100 micrometers. These miniaturized channels are often useful for handling small sized samples and allow many channels to be constructed in a small substrate, although this is not a requirement.

Often, the channel and chamber may be disposed in a solid substrate. Suitable materials for solid substrates include but are not limited to ceramics (e.g., alumina), semiconductors (e.g., silicon or gallium arsenide), glasses, quartz, metals (e.g., stainless steel or aluminum), polymers (e.g., polycarbonate, polymethylmethacrylate (PMMA), polymethylsiloxine, polydimethylsiloxane (PDMS), or polytetrafluoroethylene (Teflon^(R))), and combinations of these materials. Of course it will be appreciated that many other materials may also be used. Of these particular materials certain glasses, quartz, and polymers (e.g., PMMA and polycarbonate) are known to be substantially transparent at least in the visible spectrum. The transparency of these and other materials in the near infrared or ultraviolet is available in the literature or may easily be determined without undue experimentation. These transparent materials may be used as the substrate. Alternatively, non-transparent materials may be used as the substrate and transparent materials may be used as windows to allow sufficient transmittance of the relevant radiation. Materials that are suitable for windows include the transparent materials previously mentioned as well as other materials such as sapphire, magnesium fluoride, and calcium fluoride crystals. One exemplary substrate contains a channel and cavity formed in a stainless steel substrate, the channel and the cavity surfaces lined with an inert material such as Teflon^(R) (for improved chemical compatibility), and one or more transparent windows of these or other transparent materials formed into the chamber to allow transmittance of radiation into the chamber. Alternatively, the stainless steel substrate may be replaced with glass, quartz, or ceramic, which should have sufficient compatibility with samples that omitting the Teflon coating would be appropriate. As yet another example, it may be desirable to use a polymer to help reduce fabrication costs and take advantage of molding, hot embossing, and other fabrication techniques. An exemplary substrate may include polydimethylsiloxane containing a channel and chamber housing molded or embossed therein into which one or more windows and reflectors may be inserted and affixed.

Various micromachining methods, such as micromilling, laser ablation, and focused ion beam milling may be used to form channels in ceramic, glass quartz, semiconductor, metal, and polymeric substrates. Approaches for fabricating microfluidic channels are described for example in U.S. Pat. Nos. 5,904,824, 6,197,503, 6,379,974, 6,409,900, and 6,425,972. Additionally, photolithographic etches that are commonly used in the semiconductor processing arts may be used to form channels in semiconductor, quartz, glass, and certain ceramic and metal substrates. For example, reactive plasma may be used to etch a channel having a depth of a few hundred microns and smooth vertical sidewalls in a silicon substrate. Photolithographic etches based on different chemistries may similarly be used to form channels in polymeric materials. In addition, molding, injection molding, stamping, hot embossing, and other approaches may be used to form channels in polymeric materials, as well as certain metals. Other techniques that are suitable for forming channels, as well as further background information on microfabrication is available in “The MEMS Handbook” (ISBN/ISSN: 0849300770), by Mohamed Gad-el-Hak, published on Sep. 27, 2001, containing pages 1-1368, and available from the University of Notre Dame.

A chamber according to embodiments of the invention contains a resonant cavity to contain a sample for analysis, at least one window to the cavity to transmit a first radiation having a first frequency (e.g., an input excitation radiation from a laser) into the cavity and to transmit a second electromagnetic radiation having a second frequency (e.g., an output beam of stimulated Raman radiation) out of the cavity, and a plurality of reflectors (e.g., multi-layer dielectric mirrors) affixed to a housing of the cavity to reflect radiation of a predetermined frequency (e.g., the stimulated radiation).

Referring to FIG. 2 and the particular chamber 250, the resonant cavity is a region within the fluid channel between a reflector 235 and a partial reflector 245. The chamber has an inlet opening, at the leftmost edge of the reflectors 235, 245 (as viewed), to allow addition of a sample, and an outlet opening, at the rightmost edge of the reflectors 235, 245 (as viewed), to allow removal of the analyzed sample. The sample may either be flowed continuously through the chamber or stopped within the chamber. Alternatively, the chamber may be located at a dead-end of the channel and may utilize a common inlet-outlet opening to add and remove a sample.

Once the sample is contained within the chamber, with reference to block 120 of the method 100 (FIG. 1), the sample is irradiated, hi the system illustrated in FIG. 2, the source 210 provides input radiation 215 to the chamber and irradiates the sample positioned therein. Suitable sources include coherent light sources, lasers, light emitting diodes (LEDs), lamps, and fiber optic cables coupled with such a source of radiation. Radiation sources that provide strong monochromatic or quasimonochromatic radiation will often be favored over those that provide weaker or broad-spectrum radiation, since such monochromatic radiation facilitates detection of the relevant offset spectroscopic signals. The source 210 may contain filters or monochromators to reduce certain frequencies. The source may also contain lenses, mirrors, or other devices to redirect and focus the radiation on the chamber. These devices are commercially available from numerous sources, including vendors that are listed in “The Photonics Directory™: The Photonics Buyers' Guide To Products and Manufacturers”. This directory contains vendor listings for radiation sources, radiation detectors, spectrometers, spectroscopic analysis software, as well as numerous other accessories (e.g., lenses, wavelength selection devices, etc.). The Photonics Directory™ is available from Laurin Publishing, and is presently available online at the website website: www.photonics.com/directory/index.asp.

A laser (light amplification by stimulated emission of radiation) may be used to provide a high intensity beam of coherent and monochromatic light having wavelengths suitable for resonance enhanced Raman spectroscopy. Many different types of lasers are suitable including tunable lasers, gas lasers, solid-state lasers, semiconductor lasers, laser diodes, VCSEL (Vertical-Cavity Surface-Emitting Laser), and quantum cavity lasers. The laser may be used to provide radiation at frequencies or wavelengths that are suitable for Raman spectroscopy, including radiation in the visible, often in the green, red, or near infrared, and ultraviolet regimes. Many molecules of potential interest, including many nucleic acid derivatives, have their resonance wavelengths in or near the ultraviolet spectrum. For example, adenine, a DNA base, has a near maximum resonance wavelength at approximately 267 nanometers (nm, one billionth of a meter). As such, the ultraviolet spectrum as an excitation wavelength may provide comparatively strong signals. Potential drawbacks to using ultraviolet as excitation radiation is that depending on the intensity it may damage the molecule of interest and may induce background noise from other system components and compounds that also have resonance wavelengths in the ultraviolet. Lasers and associated optics for the ultraviolet range also tend to be more costly. Another option is the use of near infrared or visible excitation radiation. While the signals may be slightly less intense compared to ultraviolet, there may be comparatively less background noise, since most materials have their resonance wavelengths outside of these spectral frequencies. Although this particular laser is not required, the present inventors have utilized an argon-ion laser to provide a radiation at about 514 nanometers wavelength and have found that such a laser is suitable for differentiating the Raman spectra of DNA nucleotides. This particular laser is commercially available from Coherent Inc. of Santa Clara, Calif. and Spectra-Physics, Inc. of Mountain View, Calif. The laser may be operated at a sufficient intensity that allows detection and avoids damaging the molecule. As an example, the laser may be operated at energy in the range of approximately 100 W to 100 kW, depending upon the stability of the molecule. Other suitable lasers include a 10 mW helium-neon laser providing radiation at a wavelength of 633 nanometers, a linearly polarized 50 mW diode laser providing radiation in the near infrared at a wavelength of 785 nanometers, and other lasers.

Referring to FIG. 2, the chamber receives input radiation from the source through at least one window into the cavity that allows the sample positioned therein to be irradiated. The particular chamber illustrated receives transmitted radiation 225, which is transmitted through the fluid channel walls, through an inlet window 220 opening to the cavity at a left edge of the reflectors 235, 245. The inlet window may be a portion of the cavity housing that is at least partially transparent to the particular excitation radiation, a position adjacent to a reflector, a space or gap between reflectors, an incomplete or finite reflector through which a portion of the radiation may pass, a lens, an electro-optic device, or a waveguide (e.g., a fiber optic material or wispering gallery mode) that confines directs and guides the radiation into or out of the cavity. Suitable electro-optic devices include laser modulators and Pockel cells. A Pockel cell is a solid state electro-optic device that is often used as a Q-switch. An electrical field or other signal is applied to the device in order to modify or switch the birefringence of the cell. This allows modifying the transmittance of radiation through the cell and switching it on or off. Accordingly, the electro-optical device may act as a window that may be opened or closed by applying appropriate electrical current in order to allow transmittance of light through the device into and/or out of the chamber. Electro-optic devices and Pockel cells are available commercially from Conoptics, Inc. of Danbury Conn., among other vendors. It is also contemplated that an actual moving shutter may be opened and closed to allow light into and/or out of the chamber. Hinged members commonly used in MEMS (micro-electromechanical) devices may be used for the shutter.

As previously discussed, the transmitted input radiation 225 is introduced into the cavity and some of the input radiation, or some of the reflected input radiation, or both, irradiates the sample. With reference to block 130 of the method 100 of FIG. 1, some of the radiation is scattered by the sample. As used herein, the terms “scattered radiation” and the like will be used to refer to a photon of light or other radiation that has collided with and been absorbed by sample matter, such as a molecule, and has been released or emitted. Most of the scattered photons will be released elastically in which the scattered radiation has no change of energy or frequency. This radiation is known as Rayleigh scattered radiation. Some of the scattered photons, often a small fraction, will be released inelastically in which there is an exchange of energy and a change of frequency. These are well-known phenomenon. Stokes scattered radiation refers to scattering where the molecule loses energy and the frequency is reduced whereas anti-Stokes scattered radiation refers to scattering where the molecule gains energy and the frequency is increased. This scattering of light by matter is known as Raman spectroscopy. FIG. 3 conceptually illustrates Rayleigh, Stokes, and anti-Stokes radiation. Stokes and anti-Stokes radiation typically have lower intensity than Rayleigh radiation, and respectively exist at lower and higher frequencies than Rayleigh radiation. As used herein, Stokes and anti-Stokes radiation will be referred to collectively as inelastically scattered radiation.

The change in frequency or wavelength of the inelastically scattered radiation is based on the particular characteristics of the sample or molecule of interest contained therein. That is, the difference between the frequencies of the input radiation and the inelastically scattered radiation is characteristic of the sample. For example, the frequency of a photon scattered from an inelastic collision with a molecule may depend upon the polarization, vibration, rotation, and orientation characteristics of a molecule. The reasons for this are explained by quantum mechanics. Briefly, the photon may excite the molecule from its ground vibrational state to a high-energy or virtual vibrational state, from which it may relax to a lower-energy vibrational state by emission of inelastically scattered radiation, and ultimately relax back to the ground state. Further background information on this process as well as further background information on other vibrational spectroscopy topics is available in “The Handbook of Vibrational Spectroscopy”, by John Chalmers and Peter R. Griffiths, published by John Wiley & Sons, Ltd. In any event, the frequency shifts in the light that is inelastically scattered by a molecule of interest incorporate information comprising a fingerprint or signature for the molecules polarization, vibration, rotation, and orientation characteristics.

As previously discussed, it is often difficult and improbable to detect, or reliably detect, spontaneously scattered radiation from a dilute molecule in solution. Accordingly, with reference to block 140 of the method 100 of FIG. 1, the present inventors have discovered systems and methods for resonating radiation in a cavity in order to perform a resonance enhanced spectroscopic analysis of a sample. The resonated signal is stronger and easier to detect than the spontaneous signal. Advantageously, this may allow improved probability and reliability of accurately and reliably detecting and identifying dilute molecules of interest in solution.

The particular chamber 250 of FIG. 2 is a resonate chamber containing the reflector and the partial reflector to confine and resonate a radiation within the cavity in order to increase or amplify its intensity. The inelastically scattered radiation may be resonated in order to increase its intensity and aid detection and identification of a sample positioned within the chamber. The reflectors may be incorporated into or affixed to the housing of the channel to internally reflect radiation within the cavity. Radiation that is inelastically scattered by the sample or a portion of the input radiation 225 that is reflected by the reflector 235 may be reflected by the partial reflector 245 and resonated in the cavity. Portions of the radiation that are not reflected sufficiently parallel to the optical axis normal to the reflectors may leave the chamber quickly. The reflectors may have lengths that are smaller than the distance of separation between the reflectors to allow rapid removal of misaligned radiation. In this way, in addition to resonating a particular frequency or range of frequencies, the chamber effectively selects a direction of radiation that accumulates in the chamber, thereby allowing an intense, coherent beam of radiation.

The cavity may have a shape and size that facilitates resonance. As used herein, unless specified otherwise, resonance refers to the intensification of radiation in a chamber due to reflection, and is not to be confused with excited states of electrons within a molecule. As illustrated, chamber 250 has the reflectors opposite and parallel to one another, centered along a common optical axis, and separated by a particular predetermined distance that is proportional to or at least based on a wavelength of radiation to be resonated in the chamber, and that provides a non-destructive relationship between the phases of incident and reflected radiation. When the distance between the reflectors is a multiple of approximately half the wavelength of the radiation field, the partial waves may overlap substantially constructively, otherwise they may overlap less constructively or destructively. The particular reflectors may be separated by a distance that is approximately one half the product of the mode of the resonance frequency, times the speed of light, divided by the index of refraction of the medium filling the cavity, divided by the frequency of the particular radiation to be resonated in the chamber. Since coherent Raman spectroscopy may utilize excitation radiation in the visible, near infrared, and ultraviolet regimes, and since the inelastically scattered radiation is merely offset from the excitation radiation, the cavity may be designed to resonate inelastically scattered radiation having virtually any wavelength in the visible, ultraviolet, and near infrared regimes, depending upon the particular excitation radiation desired. The particular choice of excitation radiation may depend upon the cost of the laser and detectors, the capability of the detectors to detect a particular radiation, the resonance and stability of the molecule of interest, the false signals or noise caused by other absorbing molecules or species that are not of interest, etc.

A chamber may be designed to resonate an inelastically scattered radiation for a molecule of interest (e.g., a particular nucleotide) or set of molecules of interest (e.g., a set of nucleotides). As one example, consider for a moment the Stokes spectra shown in FIG. 4 (which will be discussed in more detail below) generated from excitation with a 514 nanometer argon-ion laser. The bottommost spectra for the molecule deoxythymidine monophosphate has a pronounced peak at a Stokes Raman shift of approximately 1350 cm-1. This shift is offset from the excitation wavenumber of 19455 cm⁻¹ (i.e., 10⁷/514=19455) for the 514 nanometer excitation radiation, and translates into an actual Stokes Raman wavenumber of approximately 18105 cm⁻¹ (i.e., 19455-1350). This corresponds to a wavelength of 552 nanometers (i.e., 10⁷/18105). This Stokes scattered radiation may be resonated in the chamber in order to amplify the optical signal and improve detection. The particular reflectors shown in FIG. 2 may be separated by a distance that is approximately one half the product of the mode of the resonance frequency times the speed of light divided by the index of refraction of the medium filling the cavity divided by the frequency of the particular radiation to be resonated in the chamber for this 552 nanometer scattered radiation. As another example, the chamber may be designed to resonate this wavelength of radiation as well as wavelengths of inelastically scattered radiation from other molecules of interest. In one embodiment of the invention, the predetermined distance between the reflectors is a function of wavelengths corresponding to prominent peaks in the Raman spectra of a plurality of molecules of interest (see e.g., the peaks in FIG. 4). The function may be an average, a weighted average, or other combinatorial functions.

The reflector 235 and the partial reflector 245 may be multiple layer dielectric mirrors, metal mirrors, or other reflectors that are commonly utilized in the spectroscopy, laser, optics, or fiber optic arts. A multi-layer dielectric mirror, of which a Distributed Bragg Reflector (DBR) is one example, is an interference based mirror structure containing a stack or laminate of alternating layers of materials having different indices of refraction. The thickness of the layers may be proportional to the wavelength of the radiation to be reflected such that the reflected waves from the layers are in phase with one another and superimpose. The reflector may contain alternating layers of low and high refractive index materials. A dielectric or insulating material is often used for at least one of the alternating layers and the other layer may be a different dielectric material, a non-dielectric material, a semiconductor, or other materials. One non-limiting example of a dielectric mirror includes a plurality of alternating layers of silicon (Si) and an oxide of silicon (e.g., silicon dioxide, SiO₂), for example SiO₂/Si/SiO₂ that each have a thickness approximately quarterwavelength (i.e., ¼ the wavelength) of the reflected radiation (e.g., inelastically scattered radiation). More pairs of alternating layers may be added to improve reflectance. The partial reflector 235 may have less total layers than the reflector 245 in order to transmit relatively more incident radiation than the reflector 245. Another non-limiting example of a dielectric mirror includes a quarterwavelength thickness of gallium arsenide (GaAs), a stress matched SiO₂/Si₃N₄/SiO₂ trilayer (alternating dielectric layers), and about 1500 Angstroms of gold. That is, this mirror contains both a dielectric interference based mirror as well as a metal mirror. Numerous other examples of multi-layer dielectric mirrors abound in the literature. Multi-layer dielectric mirrors may achieve high reflectance that are often in the range of approximately 99-99.9%, or higher. These mirrors may be used to rapidly achieve good gains and increased intensities.

A multi-layer dielectric mirror may contain layers that have a thickness that is proportional to a wavelength of an inelastically scattered radiation for a molecule of interest (e.g., a particular nucleotide) or set of molecules of interest (e.g., a set of nucleotides). For example, the layers may have a thickness that is approximately quarterwavelength of the average anti-Stokes or Stokes scattered radiation for a set of monobasic nucleic acid derivatives for DNA. This may allow a single resonant cavity to be used to perform resonance enhanced spectroscopic analysis of the entire set of derivatives, for example in the context of DNA sequencing, and allow identification of a particular derivative of the set based on a resonance intensified beam incorporating derivative specific frequency shift information that is transmitted from the cavity. As another example, the layers may have a thickness that is approximately quarterwavelength of the 552 nanometer Stokes scattered radiation for molecule deoxythymidine monophosphate exposed to 514 nanometer radiation from an argon-ion laser. Other thicknesses may apply for other molecules and excitation radiations.

Multi-layer dielectric mirrors are available commercially from a number of sources or may be fabricated. SuperMirrors™ are examples of suitable multi-layer dielectric mirrors that are available from Newport Corporation of Irvine, Calif. The SuperMirrors™ may be obtained and affixed to the housing of the chamber. Alternatively, a multi-layer dielectric mirror may be fabricated by techniques that are commonly used in the semiconductor processing arts (see e.g., U.S. Pat. No. 6,320,991 or 6,208,680). As another example, a dielectric mirror containing alternating layers of Si and SiO₂ may be deposited by a conventional chemical vapor deposition or physical vapor deposition technique that is commonly used in the semiconductor processing arts. For example, a low pressure chemical vapor deposition (LPCVD) technique may be used to provides good quality layers of controlled thickness. Alternatively, a physical vapor deposition technique such as evaporation, sputtering, or molecular beam epitaxy may be used to deposit the layers. As another variation, a thicker silicon layer may be deposited by a chemical vapor deposition and a silicon dioxide layer may be thermally grown from the silicon layer to a similar thickness. Of course, the chemical and physical deposition techniques may also be used to deposit other materials.

The reflector and partial reflector may alternatively be metal mirrors. Suitable metals include, among others, aluminum, gold, silver, chrome, and alloys, mixtures, or combinations thereof. Hereinafter the term metal will include pure metals as well as alloys, mixtures, or combinations. Suitable metal mirrors are available from Newport Corporation. Metal mirrors may also be fabricated by conventional chemical vapor deposition and physical vapor deposition techniques. For example, a gold layer may be deposited on a cavity wall by molecular beam epitaxy evaporative deposition. Metal mirrors may provide reflectance in the range of approximately 90-95%, or slightly higher but usually not greater than about 99%.

The reflected radiation may be resonated within the cavity and may stimulate or induce other inelastic scattering events. The stimulated events may occur more rapidly or more probabilistically than spontaneous scattering events and the intensity of the inelastically scattered radiation may build within the chamber until saturation or equilibrium is achieved wherein round trip gain due to resonance matches loses. It is anticipated that intensities up to on the order of a hundred milliwatts may be obtained. Such resonance enhanced stimulated Raman spectroscopy may provide an amplified, intense, coherent, frequency-shifted beam that should allow probable, accurate, and reliable detection and identification of even a single molecule of interest in dilute solution. It is not required that a single molecule of interest be analyzed and in other embodiments a sample containing any desired number of molecules of interest may be analyzed.

By now it should be apparent that different types of samples may be analyzed. As used herein, the term sample will refer to one or more molecules, atoms, or ions to be analyzed either alone or with a vapor, gas, solution, solid, or sorbent (e.g., a metal particle or colloidal aggregate). The sample may contain a dilute solution containing one or more molecules of interest. Virtually any molecule may be of interest, depending on the particular implementation, such as a biologic molecule, a molecule associated with nucleic acid sequencing, a pharmaceutical, pesticide, an herbicide, a polymer, a pollutant, or others. For example in the case of analyzing a biological molecule of interest associated with nucleic acid sequencing, an aqueous or other solution containing a protein, protein fragment, or nucleic acid derivative may be analyzed. The term nucleic acid derivative will be used to refer to a nucleic acid, nucleic acid fragment, nucleotide, nucleoside (e.g. adenosine, cytidine, guanosine, thymidine, uridine), base (e.g. adenine, cytosine, guanine, thymine, uracil), purine, pyrimidine, or a derivative of one of these molecules. One exemplary sample contains a solution of a single nucleic acid derivative having a single base that is selected from the group consisting of adenine, cytosine, guanine, thymine, and uracil.

Proteins provide a number of critical reactions, structures, and controls for cells. Nucleic acids provide cells information about which proteins to synthesize. Nucleic acids are linear polymers of nucleotides connected by phosphodiester bonds. Nucleic acids may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Nucleotides consist of three parts, a phosphate group, a pentose (a five carbon sugar molecule), and a base. Nucleosides are bases and sugars without a phosphate group. The bases of both nucleotides and nucleosides are adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U). The bases adenine, cytosine, and guanine are found in both DNA and RNA, thymine usually found only in DNA, and uracil is usually found only in RNA. Accordingly, the nucleosides for DNA include deoxyadenosine, deoxyguanosine, deoxycytidine, and deoxythymidine, whereas the nucleosides for RNA include adenosine, guanosine, cytidine, and uridine. Likewise, the nucleotides for DNA include deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxycytidine monophosphate, deoxythymidine monophosphate (or in the salt form, deoxyadenylate, deoxyguanylate, deoxycytidylate, and thymidylate), whereas the nucleotides for RNA include adenosine monophosphate, guanosine monophosphate, cytidine monophosphate, uridine monophosphate (or in the salt form, adenylate, guanylate, cytidylate, and uridylate). In RNA the pentose is ribose whereas in DNA the pentose is deoxyribose. The bases cytosine, thymine, and uracil are pyrimidine bases and contain a single heterocyclic ring containing carbon and other atoms, in this case nitrogen. The bases adenine and guanine are purine bases and contain a pair of fused heterocyclic rings that also contain nitrogen. The ability to determine a nucleic acid and/or protein sequence may have tremendous commercial importance in the fields of medical diagnosis, treatment of disease, and drug discovery. Of course, these are just examples of the types of samples and molecules that may be analyzed by the systems and methods developed by the present inventors.

With continued reference to the method 100 of FIG. 1, and to block 150, after resonating, the scattered radiation may be irradiated or transmitted from the chamber. Referring to FIG. 2, the chamber may contain at least one window to allow the radiation to exit the chamber and be detected. The particular chamber 250 contains an outlet window 260, which in this particular instance comprises the partial reflector 245. The partial reflector is an incomplete or finite reflector that has a sufficient reflectivity to achieve resonance and a sufficient transmittance to allow a detectable level of incident radiation to be transmitted to the detector 290. The reflector 235 may have a higher total reflectance than the partial reflector 245 in order to provide good amplification of intensity in the cavity, although this is not required. In one instance, the reflector 235 may have a high reflectivity for both the input excitation radiation and the inelastically scattered radiation, for example greater than approximately 99% (e.g., approximately 99.9%) for both, whereas the partial reflector 245 may have a sufficient transmittance for the inelastically scattered radiation, for example a transmittance that is not less than approximately 1% (or a reflectance that is not greater than approximately 99%). Of course alternate types of outlet windows may also be used, such as a transparent material incorporated into the cavity housing, a Pockel cell, a space, gap, slit, pinhole, or other opening in the reflector 235, or a shutter. Additionally, as another alternative, a single inlet-outlet window may be utilized to transmit radiation into and out of the chamber.

At least a portion of the resonated radiation 240 that is incident on the partial reflector 245 leaves the cavity and chamber as output radiation 285. The output radiation contains inelastically scattered radiation that incorporates frequency-shift information about the sample in the chamber. The intensity of the output radiation may depend upon the reflectivity of the partial reflector, the resonance characteristics of the chamber, and upon losses due to absorption, scattering and diffraction. Often, the chamber will be designed to achieve high intensities of the output radiation to allow accurate detection and identification of samples.

With reference again to the method 100 of FIG. 1, at least some of the inelastically scattered radiation in the output radiation 285 is detected at block 160. The electromagnetic radiation detector 290 is shown in simplified format and is to be interpreted broadly. Often, the output radiation may be passed through a lens and a wavelength selection device. The lens may collect, direct, and focus the radiation emitted from the chamber. The light from the lens or from the chamber may be passed through a wavelength selection device that emphasizes, selects, separates, or isolates a particular frequency or range of frequencies. The wavelength selector may be used to distinguish radiation of interest (e.g., inelastically scattered radiation) from other radiation (e.g., elastically scattered radiation and excitation radiation). Suitable wavelength selection devices include among others prisms, monochromators, filters (e.g., absorbance, bandpass, interference, or Fourier), dichroic filters, dichroic mirrors, and demultiplexers.

After passing the radiation from the chamber through any lenses, wavelength selection devices, and the like, the resultant radiation may be passed to a spectrometer, optical multichannel analyzer (OMA), or other radiation detection device. These radiation detection devices will often employ an optical transducer to convert received radiation into a corresponding electrical signal that captures and represents at least some of the same information. Exemplary optical transducers include a charge coupled device (CCD), phototransistor, photomultiplier tube, photo diode, or an array of one or more of these devices.

The radiation detection device may comprise a Raman spectrometer. Spectrometers are well known radiation detection devices that measure the wavelength and intensity of light. Spectrometers are commercially available from a number of sources. One suitable Raman spectrometer is SpectraPro 300i Spectometer available from Acton Research Corporation of Acton Mass. Another spectrometer that is suitable is the RAMANRXN1 Analyzer available from Kaiser Optical Systems, Inc. (website: www.kosi.com/) of Ann Arbor, Mich. This spectrometer uses a thermoelectrically cooled Charge Coupled Device (CCD) array to provide high sensitivity detection of radiation. Optionally, this spectrometer may also be obtained with an Invictus NIR Laser as a radiation source, a SuperNotch® Filter, a Holographic Laser Bandpass Filter, and a HoloSpec holographic imaging spectrograph. Further background information about this spectrometer including its operation is available from the vendor. Of course other spectrometers may also be used.

Often, the spectrometer may generate spectra showing frequency or wavelength versus intensity. FIG. 4 shows Stokes Raman spectra for highly diluted aqueous samples of each of the four DNA nucleotides, according to embodiments of the invention. For clarity, the four spectra have been offset or displaced from one another along the vertical axis. From top to bottom, the spectra are for deoxyadenosine monophosphate, deoxycytidine monophosphate, deoxyguanosine monophosphate, and deoxythymidine monophosphate. The spectra were generated by performing spontaneous Raman spectroscopic analysis on the samples including irradiating the samples with 514 nanometer radiation from a laser and detecting the output inelastically scattered radiation with a Raman spectrometer. The spontaneous spectra should be substantially similar to resonance enhanced Raman spectra except that the signals would be correspondingly weaker in intensity. The figure shows intensity in arbitrary units versus Raman wavelength shift (Stokes offset from the excitation radiation) in reciprocal centimeters. As shown, each of the spectra have distinct spectral characteristics, prominent peaks, or fingerprint bands, that identify the corresponding nucleotide. Exemplary prominent peaks include 1630 cm⁻¹ for the top spectra, 1440 cm⁻¹ for the next spectra down, 800 cm⁻¹ for the second spectra from the bottom, and 1350 cm⁻¹ for the bottom spectra.

Finally, with reference to the method 100 of FIG. 1, the sample may be identified at block 170. The sample may be identified by comparing the determined spectrum with a spectral library containing many predetermined spectrum for known samples and identifying the sample as one of the known samples if the determined spectrum sufficiently approximates the corresponding predetermined spectrum in the library. The electrical signals generated by the radiation detector as a result of the output radiation may be provided to a computer system that is appropriately programmed with spectroscopic analysis instructions and the library (e.g., a database) that allows the Raman fingerprint or signature represented in the electrical signals to be correlated to a specific fingerprint or signature in the library. For example, in the case of a spectrometer, a spectrum (or certain bands thereof) for a sample may be compared or contrasted to spectroscopic data for other identified samples to determine whether the fingerprints are sufficiently identical (i.e., the sample has the same identity as the identified sample in the database). Various methods for identification of nucleotides by Raman spectroscopy are known in the art (see e.g., U.S. Pat. No. 5,306,403, 6,002,471, or 6,174,677). The identity of the sample may be stored in memory, further analyzed, or used for other purposes.

With reference again to the method 100 of FIG. 1, another way of implementing this method may include adding a sample to a chamber at block 110, irradiating the sample with a short and accurately-known pulse of a radiation having substantially the same frequency or wavelength as an inelastically scattered wavelength relevant to the sample at block 120, then scattering, resonating, and irradiating the radiation from the cavity at blocks 130-150, respectively, and then detecting the radiation as well as some indication of the decay time of the inelastically scattered light in the chamber at block 160. The decay time may be used as a fingerprint or signature for the identity of the sample. Of course other methods are also contemplated.

FIG. 5 shows a cross-sectional view of a spectroscopic analysis system 500 suitable to perform a resonance enhanced spectroscopic analysis of a sample in order to identify the sample, according to embodiments of the invention. The system includes a radiation source 510, a substrate 505, a fluid channel 570, a spectroscopic analysis chamber 550, and a radiation detector 590. A sample 530 is contained within the chamber. The channel and the chamber are formed within the substrate and are coupled. The particular chamber contains a spheroidal resonant cavity, an inlet window 520, a first, second, third and fourth curved reflectors 535A-D, and an outlet window 560. The sample may be added to the channel at an inlet 575 thereof, then flowed into a cavity of the chamber and spectroscopically analyzed, and then flowed out of the chamber and through the sample outlet 580.

The spheroidal resonant cavity is formed of concave surfaces that may assist with reflecting radiation toward the interior of the cavity. The term spheroidal will be used to refer to a shape that resembles or approximates a sphere but that is not necessarily a perfect sphere. Of course, the spheroidal shape is not required and in other cavities only a portion of the chamber housing may be concave (e.g., spherically or parabolically concave), or the cavity may be a cylindrical void (e.g., having a diameter greater than that of the fluid channel), a polyhedron void, a hexahedron void, a cubic void, or a void having any other regular or irregular shape.

The cavity serves as a wide spot in the fluid channel and may have a volume that is convenient for the particular implementation. Typically, the cavity will have a volume that is not greater than about a milliliter (mL, one-thousandth of a liter). Alternatively, if a smaller cavity is favored, the cavity may be a micro-sized cavity or microcavity having a volume that is not greater than approximately a microliter (μL, one-millionth of a liter). For example, the volume may be approximately 500 nanoliters (nL, one billionth of a liter), approximately 100 mL, approximately 1 nL, or less.

An input excitation radiation 515 from the source 510 is directed through the substrate, through the inlet window 520, and into the cavity. The particular inlet window 520 comprises a region of the cavity housing between the second and third reflectors 535B-C that is at least partially transparent to the inlet radiation 515. The inlet window may be a space between the second and third reflectors, for example a portion of sufficiently transparent substrate, or a partial reflector between the second and third reflectors or a transparent material. At least some of the input radiation irradiates the sample and is inelastically scattered. The reflectors 535A-D confine the inelastically scattered radiation, and in this particular embodiment much of the input radiation, inside the chamber by internal reflection. Some of the reflected radiation may stimulate further inelastically scattered radiation by re-irradiating the sample and again being inelastically scattered. In another embodiment of the invention, such as that discussed in FIG. 8, an outlet window may be used to remove the input excitation radiation from the cavity.

Output radiation 585 leaves the cavity through the outlet window 560 and contains at least some resonance enhanced stimulated inelastically scattered radiation. The outlet window is positioned away from the path of the input radiation, and primary reflections thereof, to reduce transmission of the inlet radiation through the outlet window. Of course, this is not required, and a wavelength selection device may be used to remove any such radiation that is transmitted.

FIG. 6 shows another cross-sectional view of a spheroidal chamber 650, according to embodiments of the invention. The chamber shows the features of the chamber 550 as well as alternate locations 676, 677, 678, and 679 for either the inlet window 620 or outlet window 660. By way of example, the outlet window may be re-positioned at one of the locations 676-679, or the inlet window may be re-positioned at one of the locations 676-679. A sample 630 is positioned within the chamber. FIG. 6 also shows section lines 7A-7A and 7B-7B that respectively show the views of FIGS. 7A and 7B.

FIGS. 7A-7B show fabrication of the chamber 650 of FIG. 6, according to embodiments of the invention. FIG. 7A shows a top-plan view of the top of the chamber 650 along the section lines 7A-7A shown in FIG. 6. The hemi-spheroid void 750A of the top of the chamber 650 and the hemi-cylindrical voids 770A (half of a cylindrical channel void) may be formed within a substrate 705A. In one example, these voids 750A, 770A may be formed in a quartz or glass substrate by photolithography and etching. An anisotropic etch with agitation may be used to form the nearly spherical concave surfaces of the chamber 750A and the concave cylindrical surfaces of the channel 770A by etching along crystal planes. In another example, these voids may be formed in a polymeric material, such as PDMS by molding or embossing.

The interior surface of the hemi-spheroid void 750A contains reflectors 735B-D (which correspond to the reflectors 635B-D in the view of FIG. 6). The reflector may be formed by subsequent alternating deposition of layers of appropriate thickness of materials having different index of refraction (e.g., Si and SiO₂). The depositions may be over the whole hemisphere in which case an inlet window 720 and an exit window 760 may be formed by removing a portion of these deposited layers to form a partial reflector, or by removing all of these layers to form a transparent quartz window. These layers may be removed by laser ablation, by etching, or by other conventional techniques for removing layers. Alternatively, a mask patterned with the sizes and positions of the windows 720 and 760 may be used to selectively deposit the multi-layer dielectric mirror layers over the non-window portions. In the case of the PDMS polymer, a sapphire, quartz, or other transparent window may be molded into the polymer or the polymer drilled and the window fixedly inserted.

FIG. 7B shows a top-plan view of the bottom of the chamber 650 along the section lines 7B-7B shown in FIG. 6. The hemi-spheroid void 750B of the bottom of the chamber 650, the hemi-cylindrical voids 770B, and the reflector 735B may be formed within a substrate 705B, such as a silicon substrate, as described for FIG. 7A. After forming these structures, the substrate 705A may be bonded to the substrate 705B by a wafer bonding approach, for example by a high temperature fusion of oxidized surfaces thereof, or with an adhesive.

FIG. 8 shows a top-plan view of a spectroscopic analysis chamber 850 configured to receive input excitation radiation in the form of aligned seed radiation 815B and transverse radiation 815A, according to embodiments of the invention. The input transverse excitation radiation may have any suitable frequency. The direction of the transverse radiation is crosswise, perpendicular, to the direction of the output radiation 885, and to the direction of resonance. This allows a portion of the transverse radiation 886 that does not irradiate a molecule 831, to leave the chamber. This may aid in detecting the output inelastically scattered radiation 885. The seed radiation is added to the chamber through a partial reflector 845. The seed radiation may have the same frequency as radiation inelastically scattered from a particular sample and may stimulate scattering if that sample is contained within the chamber. The direction of the seed radiation is opposite, but aligned with, the direction of output inelastically scattered radiation 885. This allows the seed radiation transmitted through the partial reflector to be reflected and resonated. The radiation detector may be configured to determine the gain or increase in intensity of the output radiation over the known intensity of the input seed radiation, which gain or increase may be attributed to stimulated scattering.

Accordingly, with continued reference to the method 100 of FIG. 1, irradiating a sample at block 110 may include irradiating a sample with an input transverse radiation and an input seed radiation having different frequencies. Likewise, detecting radiation at block 160 may include detecting a gain over the intensity of the seed radiation. The seed radiation frequency may be varied over a set of predetermined frequencies corresponding to different molecules to determine whether the sample within the chamber contains a molecule corresponding to one of these frequencies. For example, the seed radiation may be provided at an inelastically scattering frequency for adenine. If the gain over the seed radiation is sufficiently zero then there was no simulated scattering and it may be concluded that the sample did not contain adenine. Then the frequency of the seed radiation may be adjusted to that corresponding to other nucleotide bases until the gain is sufficiently nonzero indicating that the sample may contain that particular base. If none of the frequencies cause the gain to become sufficiently nonzero, the sample may be removed from the chamber and another sample added thereto.

The concepts of seed and transverse input radiations discussed in FIG. 8 may also be applied to a spectroscopic analysis system similar to the ones shown in FIGS. 5-6. For example, input transverse excitation radiation may be transmitted in through window 620, input seed radiation may be transmitted in through window 660, and scattered radiation may be transmitted out through window 660. Portions of the input transverse radiation that do not irradiate the sample may be removed through a window at location 679.

Accordingly, in embodiments of the invention employing a stimulated Raman analysis, a single molecule of interest may be positioned within a resonant chamber that is capable of optically amplifying the intensity of inelastically scattered stimulated Raman radiation by causing resonance enhanced stimulated scattering events that aid in increasing or amplifying the intensity of the signal to an extent that allows detection and accurate identification of the single molecule of interest. As an example, a single nucleic acid derivative of interest may be positioned in a resonant chamber having two opposing reflectors. The first reflector has a high reflectivity for both an excitation radiation and an inelastically scattered Raman radiation, while the second reflector has a high reflectivity for the excitation radiation and has a sufficient transmittance for the inelastically scattered Raman radiation. In one aspect, a high reflectivity may be greater than approximately 99% (e.g., 99.5%-99.9%) while a sufficient transmittance may be a reflectivity not greater than 99%. The two opposing reflectors are separated by a distance that is sufficient to resonate the inelastically scattered Raman radiation. The distance may be determined based on inelastically scattered Raman radiation for the single nucleic acid derivative, or for a plurality of nucleic acid derivatives so that the chamber may be used for identifying different derivatives.

Accordingly, a sample containing a single molecule of interest may be analyzed with stimulated Raman spectroscopy. In addition to stimulated Raman spectroscopy, another suitable form of coherent Raman spectroscopy is Coherent Anti-Stokes Raman Spectroscopy (CARS), which employs a coherent anti-Stokes Raman scattering phenomenon. Many other forms of Raman, such as spontaneous Raman and surface enhanced Raman spectroscopy (SERS), involve spontaneous emission of random and non-coherent inelastically scattered radiation. In contrast, CARS inherently produces a highly directional and coherent output inelastically scattered radiation signal. Rather than due to resonance, as in stimulated Raman, the coherency is provided by coherent build up of amplitudes for phase matched radiations. As used herein, CARS will encompass various implementations of CARS, such as time-resolved CARS, scanned CARS, OPO CARS, MAD CARS, PARS, and others. The CARS radiation signal is sufficiently coherent and intense to allow detection of even a single molecule of interest. The spectral shape and intensity of the CARS output signal contains the spectral characteristics of the sample and may be used to identify the sample. Accordingly, the present inventors contemplate employing CARS to analyze and identify a single nucleic acid derivative in conjunction with nucleic acid sequencing.

CARS is a four-wave mixing spectroscopy. During CARS a sample in a chamber is irradiated and excited with a first radiation having a first wavelength (w₁) and often a second radiation having a second wavelength (W₂) that overlap and are phase matched (i.e., they spatially and temporally overlap) in order to induce the sample to irradiate coherent anti-Stokes scattered radiation having a third wavelength (W₃) that is related to the first and second frequencies according to the relationship, W₃=2w₁−W₂.

FIG. 9 shows an energy diagram for a CARS process used to analyze a molecule of a sample, according to embodiments of the invention. Energy of the molecule is plotted on the y-axis and the progression of the CARS process is plotted on the x-axis. For convenience, the discussion will refer to a photon, although often in practice a radiation containing at least one but often many photons will be used to analyze the sample. Initially, the molecule is excited coherently with a first photon at wavelength (w₁) 910 in order to induce an emission of a second photon at wavelength (w2) 920. The first photon is often provided as a laser beam pulse directed at the chamber to irradiate the molecule. As shown, the first photon excites the molecule from a ground vibrational state 950 to a lower virtual state 970. The emitted second photon brings the molecule from the virtual state 870 down to a lower excited vibrational state 960. The excited vibrational state differs from the ground vibrational state by vibratory transition of Raman shift (W₄). The molecule may emit the second photon by either spontaneous or stimulated emission however coherent stimulated emission is used in CARS. Often, the molecule will be exposed by another photon also of wavelength (W₂) in order to induce stimulated emission of the second photon. Stimulated emission is relatively more probable than spontaneous emission and may lead to higher CARS signal intensities that facilitate detection and molecule identification. The stimulated emission is often induced by irradiating the chamber with a second radiation having the second wavelength (W₂), concurrently with the irradiation with the first radiation (the first photon).

The molecule in the lower excited vibrational state 960 is subsequently irradiated with a third photon at wavelength (w₁) 930. Irradiation with a laser beam pulse may also provide this photon. The third photon excites the molecule from the excited vibrational state 960 to a higher virtual state 980. The molecular vibrations oscillate in phase and interfere constructively. The molecule emits a fourth photon at wavelength (W₃) to drop from the higher virtual state to the ground vibrational state. The wavelength of the fourth photon is twice the wavelength of the first and third photons minus the wavelength of the emitted second photon (i.e., w₃=2w1−w₂). In essence, CARS disproportionates or divides two (w₁) photons into a (w₂) photon and a (W₃) photon. A phase matching condition determines the direction of the coherent CARS signal. As will be discussed further below, the present inventors contemplate employing various geometric configurations of beams to take advantage of this property to help spatially isolate the coherent CARS signal from input excitation beams, in order to facilitate detection and identification of molecules. The intensity of the emitted CARS signal is directly related to the intensity of the excitation radiations.

Different ways of exposing the sample to these photons are known in the arts. In one approach, the sample may be concurrently irradiated with a first laser beam pulse of the wavelength w₁, a second laser beam pulse of the wavelength W₂, and a third laser beam pulse of the wavelength (w₁). In practice, the first laser beam pulse can also serve as a third laser beam pulse as they have the same wavelength, (w₁). Electronics to provide concurrent laser beam pulses from multiple lasers are commercially available. Such synchronization electronics include SynchroLock from Coherent (Santa Clara, Calif.) or Lok-to-Clok from Spectra-Physics (Mountain View, Calif.). Alternatively, in another approach the sample may be continually irradiated with a laser beam of the wavelength (w₁) and intermittently irradiated with a pulse or scan of a laser beam of the wavelength (w₂). When the difference (w₁−w₂) substantially coincides or matches the wavelength of a molecular vibration of the sample, for example the vibratory transition (w₄), the intensity of the CARS signal is often significantly enhanced. A spectrum may be generated for a range of the wavelength (w₂) or the difference (w₁−w₂). Often, (w₂) is continually changed, varied, or scanned, while holding (w₁) fixed, and simultaneously recording the corresponding CARS signals. Alternatively, (w₁) is continually changed, varied, or scanned, while holding (w₂) fixed, and simultaneously recording the corresponding CARS signals. In another embodiment, both (w₁) and (w₂) are continually changed, varied, or scanned, to obtain the desired (w₁−w₂), while recording the corresponding CARS signals. In one approach a tunable broadband dye laser is scanned over the desired range of wavelength. Another approach involves using a broadband Stokes laser covering the whole range of frequencies concurrently. Alternatively, signal and idler beams from optical parametric oscillator can be used to scan (w₁−w₂).

In order for a CARS process to occur efficiently energy and momentum conservation need to occur. During CARS the output signal is built by coherent addition of amplitudes. In the CARS four-wave mixing process, it will often be desirable to impose a phase matching condition on the wave vectors of the input excitation radiations and output coherent anti-Stokes radiation in order to achieve good intensities of the output coherent radiation. The amplitudes should be substantially in phase, that is the beams aligned to provide phase matching between the sum of two incoming waves at wavelength (w₁) and the sum of output waves at wavelength (w₂) and (w₃). When the phase matching condition is satisfied, the strength or intensity of the output anti-Stokes wave should be near maximum. This results in the sample irradiating or emitting a coherent resonance enhanced anti-Stokes scattered radiation, often as a coherent, collimated, highly-directional beam with a predetermined direction, rather than as a weak and randomly scattered signal as would be expected in spontaneous Raman spectroscopy. The predetermined direction of the CARS signal may allow various configurations that allow it to be geometrically or configurationally separated from the excitation radiation.

A number of prior art phase matching beam configurations or geometries are known in the arts including a collinear CARS geometry, a BOX CARS geometry, a Folded BOX CARS geometry, and a USED CARS geometry. Any of these may potentially be used. Additionally, the inventors contemplate using configurations that help separate or isolate the CARS radiation signal from input excitation radiation. FIGS. 11-13 show exemplary configurations of excitation radiation beams that when phase matched may help to spatially separate and isolate the CARS signal from the excitation beams at the radiation detection end, according to various embodiments of the invention. Advantageously, in these exemplary configurations, the CARS signal beam is spatially separated and isolated from the excitation beams at the radiation detection end, which may facilitate sample identification. Other configurations will be apparent to those having an ordinary level of skill in the art and the benefit of the present teachings.

FIG. 10 shows a cross-beam configuration, according to embodiments of the invention. In the cross-beam configuration, an input first (w₁) and second radiation (w₂) are provided to a chamber 1050 with an angle relative to one another so that they irradiate a sample 1030 within the chamber and subsequently separate from one anther and from a coherent anti-stokes scattered radiation (w₃) that is irradiated from the sample. In this way, the configuration helps separate the input radiation from the output coherent scattered radiation, and wavelength selection or filtering may be avoided. The chamber may be similar to other chambers disclosed herein. The chamber may, but need not, be a resonant chamber and may, but need not, contain reflectors or have a size or shape to facilitate resonance.

FIG. 11 shows momentum conservation for a cross-beam configuration similar to that shown in FIG. 10. The phase matching condition determines the direction of the coherent anti-Stokes radiation. Because of the conservation of energy and momentum in a four-wave mixing process, a phase matching condition is imposed on the wave vectors of the input and anti-Stokes output waves. When the phase matching condition is sufficiently satisfied the output strength is near maximum and the direction and momentum of the coherent anti-Stokes photons are related to the direction and momentum of the excitation photons. This is shown graphically in FIG. 11 wherein two vectors for the first excitation wavelength (2k_(w1)) and one vector for the second excitation wavelength (k_(w2)) are combined to give a vector for the coherent anti-Stokes radiation (k_(w3)), or mathematically k_(w3)=2k_(w1)−k_(w2). The length of the vectors represents the photons momentum. The configuration of the direction of the excitation radiations allows the CARS signal photon to be geometrically separated from the input excitation photons. This may facilitate detection of the emitted CARS photon.

FIG. 12 shows a forward-scattered configuration, according to embodiments of the invention. In this configuration, the coherent radiation is not separated from the input radiations, and a wavelength selection device, such as a filter, may be used to isolate the coherent radiation.

FIG. 13 shows a back-scattered configuration, according to embodiments of the invention. In this configuration, as in the cross-beam configuration, the coherent radiation is separated from the input radiations. The intensity of the coherent radiation in this configuration is often comparable with that of the forward-scattered configuration when the sample size is less than the excitation wavelength, although in other instances it may be desirable to use a forward-scattered configuration and appropriate wavelength selection device to obtain a stronger signal.

FIG. 14 shows a nucleic acid sequencing system 1401 in which embodiments of the invention may be implemented. A nucleic acid may be provided to a sampling system 1402. The sampling system may include enzymes or other cleavers of nucleic acids to remove or derive a single nucleic acid derivative from the nucleic acid. In one instance, the system may include an exonuclease enzyme that breaks down a nucleic acid chain by removing single nucleotides one by one from the end of a chain. The sampling system may also include equipment to prepare the sample, for example to dilute the nucleic acid derivative in an aqueous solution and supply any additives. The sample may be added to the spectroscopic analysis system 1400, for example though a fluid channel 1470. The analysis system performs a Raman spectroscopic analysis of the sample, as described elsewhere herein, in order to identify the nucleic acid derivative. For example, the analysis system may determine whether the sample contains a single base selected from the group comprising adenine, cytosine, guanine, thymine, and uracil. The identity of the nucleic acid derivative may be stored in a memory as part of an ordered sequence for the input nucleic acid, analyzed as part of the sequence to solve a medical problem, for example to diagnose or treat a disease, or used for other purposes.

Having been generally described, the following examples are given as particular embodiments of the invention, to illustrate some of the properties and demonstrate the practical advantages thereof, and to allow one skilled in the art to utilize the invention. It is understood that these examples are to be construed as merely illustrative.

EXAMPLES Example 1 Nucleic Acid Sequencing Using Raman Detection and Nanoparticles

Certain embodiments of the invention, exemplified in FIG. 15, involve sequencing of one or more single-stranded nucleic acid molecules 1509 that may be attached to an immobilization surface in a reaction chamber 1501. The reaction chamber 1501 may contain one or more exonucleases that sequentially remove one nucleotide 1510 at a time from the unattached end of the nucleic acid molecule 1509.

As the nucleotides 1510 are released, they may move down a microfluidic channel 1502 and into a nanochannel 1503 or microchannel 1503, past a detection unit. The detection unit may comprise an excitation source 1506, such as a laser, that emits an excitatory beam. The excitatory beam may interact with the released nucleotides 1510 so that electrons are excited to a higher energy state. The Raman emission spectrum that results from the return of the electrons to a lower energy state may be detected by a Raman spectroscopic detector 1507, such as a spectrometer, a monochromator or a charge coupled device (CCD), such as a CCD camera.

The excitation source 1506 and detector 1507 may be arranged so that nucleotides 1510 are excited and detected as they pass through a region of nanoparticles 1511 in a nanochannel 1503 or microchannel 1503. The nanoparticles 1511 may be cross-linked to form “hot spots” for Raman detection. By passing the nucleotides 1510 through the nanoparticle 1511 hot spots, the sensitivity of Raman detection may be increased by many orders of magnitude. Alternatively, the nucleotides may be passed into a resonant chamber.

Preparation of Reaction Chamber, Microfluidic Channel and Microchannel

Borofloat glass wafers (Precision Glass & Optics, Santa Ana, Calif.) may be pre-etched for a short period in concentrated HF (hydrofluoric acid) and cleaned before deposition of an amorphous silicon sacrificial layer in a plasma-enhanced chemical vapor deposition (PECVD) system (PEII-A, Technics West, San Jose, Calif.). Wafers may be primed with hexamethyldisilazane (HMDS), spin-coated with photoresist (Shipley 1818, Marlborough, Mass.) and soft-baked. A contact mask aligner (Quintel Corp. San Jose, Calif.) may be used to expose the photoresist layer with one or more mask designs, and the exposed photoresist may be removed using a mixture of Microposit developer concentrate (Shipley) and water. Developed wafers may be hard-baked and the exposed amorphous silicon removed using CF₄ (carbon tetrafluoride) plasma in a PECVD reactor. Wafers may be chemically etched with concentrated HF to produce the reaction chamber 1501, microfluidic channel 1502 and microchannel 1503. The remaining photoresist may be stripped and the amorphous silicon may be removed.

Nanochannels 1503 may be formed by a variation of this protocol. Standard photolithography may be used to form the micron scale features of the integrated chip. A thin layer of resist may be coated onto the chip. An atomic force microscopy/scanning tunneling probe tip may be used to remove a 5 to 10 nm wide strip of resist from the chip surface. The chip may be briefly etched with dilute HF to produce a nanometer scale groove on the chip surface. In the present non-limiting example, a channel 1503 with a diameter of between 500 nm and 1 μm may be prepared.

Access holes may be drilled into the etched wafers with a diamond drill bit (Crystalite, Westerville, Ohio). A finished chip may be prepared by thermally bonding two complementary etched and drilled plates to each other in a programmable vacuum furnace (Centurion VPM, J. M. Ney, Yucaipa, Calif.). Alternative exemplary methods for fabrication of a chip incorporating a reaction chamber 1501, microfluidic channel 1502 and nanochannel 1503 or microchannel 1503 are disclosed in U.S. Pat. Nos. 5,867,266 and 6,214,246. A nylon filter with a molecular weight cutoff of 2,500 daltons may be inserted between the reaction chamber 1501 and the microfluidic channel 1502 to prevent exonuclease and/or nucleic acid 1509 from leaving the reaction chamber 1501.

Nanoparticle Preparation

Silver nanoparticles 1511 may be prepared according to Lee and Meisel (J. Phys. Chem. 86:3391-3395, 1982). Gold nanoparticles 1511 may be purchased from Polysciences, Inc. (Warrington, Pa.), Nanoprobes, Inc. (Yaphank, N.Y.) or Ted-pella Inc. (Redding, Calif.). In a non-limiting example, 60 nm gold nanoparticles 1511 may be used. The skilled artisan will realize that other sized nanoparticles 1511, such as 5, 10, or 20 nm, may also be used.

Gold nanoparticles 1511 may be reacted with alkane dithiols, with chain lengths ranging from 5 nm to 50 nm. The linker compounds may contain thiol groups at both ends of the alkane to react with gold nanoparticles 1511. An excess of nanoparticles 1511 to linker compounds may be used and the linker compounds slowly added to the nanoparticles 1511 to avoid formation of large nanoparticle aggregates. After incubation for two hours at room temperature, nanoparticle 1511 aggregates may be separated from single nanoparticles 1511 by ultracentrifugation in 1 M sucrose. Electron microscopy reveals that aggregates prepared by this method contain from two to six nanoparticles 1511 per aggregate. The aggregated nanoparticles 1511 may be loaded into a microchannel 1503 by microfluidic flow. A constriction or filter at the end of the microchannel 1503 may be used to hold the nanoparticle aggregates 1511 in place.

Nucleic Acid Preparation and Exonuclease Treatment

Human chromosomal DNA may be purified according to Sambrook et al. (1989). Following digestion with Bam H1, the genomic DNA fragments may be inserted into the multiple cloning site of the pBluescript® II phagemid vector (Stratagene, Inc., La Jolla, Calif.) and grown up in E. coli. After plating on ampicillin-containing agarose plates a single colony may be selected and grown up for sequencing. Single-stranded DNA copies of the genomic DNA insert may be rescued by co-infection with helper phage. After digestion in a solution of proteinase K:sodium dodecyl sulphate (SDS), the DNA may be phenol extracted and then precipitated by addition of sodium acetate (pH 6.5, about 0.3 M) and 0.8 volumes of 2-propanol. The DNA containing pellet may be resuspended in Tris-EDTA buffer and stored at −20° C. until use.

M13 forward primers complementary to the known pBluescript® sequence, located next to the genomic DNA insert, may be purchased from Midland Certified Reagent Company (Midland, Tex.). The primers may be covalently modified to contain a biotin moiety attached to the 5′ end of the oligonucleotide. The biotin group may be covalently linked to the 5′-phosphate of the primer via a (CH₂)₆ spacer. Biotin-labeled primers may be allowed to hybridize to the ssDNA template molecules prepared from the pBluescript® vector. The primer-template complexes may be attached to streptavidine coated beads according to Done et al. (Bioimaging 5: 139-152, 1997). At appropriate DNA dilutions, a single primer-template complex is attached to a single bead. A bead containing a single primer-template complex may be inserted into the reaction chamber 1501 of a sequencing apparatus 1500.

The primer-template may be incubated with modified T7 DNA polymerase (United States Biochemical Corp., Cleveland, Ohio). The reaction mixture may contain unlabeled deoxyadenosine-5′ triphosphate (dATP) and deoxyguanosine-5′-triphosphate (dGTP), digoxigenin-labeled deoxyuridine-5-triphosphate (digoxigenin-dUTP) and rhodamine-labeled deoxycytidine-5′-triphosphate (rhodamine-dCTP). The polymerization reaction may be allowed to proceed for 2 hours at 37° C. After synthesis of the digoxigenin and rhodamine labeled nucleic acid, the template strand may be separated from the labeled nucleic acid, and the template strand, DNA polymerase and unincorporated nucleotides washed out of the reaction chamber 1501. Alternatively, all deoxynucleoside triphosphates used for polymerization may be unlabeled. In other alternatives, single stranded nucleic acids may be directly sequenced without polymerization of a complementary strand.

Exonuclease activity may be initiated by addition of exonuclease m to the reaction chamber 1501. The reaction mixture may be maintained at pH 8.0 and 37° C. As nucleotides 1510 are released from the 3′ end of the nucleic acid, they may be transported by microfluidic flow down the microfluidic channel 1502. At the entrance to the microchannel 1503, an electrical potential gradient created by a pair of electrodes 1504, 1505 may be used to drive the nucleotides 1510 out of the microfluidic channel 1502 and into the microchannel 1503. As the nucleotides 1510 pass through the nanoparticles 1511, or alternatively into a resonant chamber, they may be exposed to excitatory radiation from a laser 1506. Raman emission spectra may be detected by the Raman detector 1507 as disclosed below.

Raman Detection of Nucleotides

A Raman detection unit as disclosed in Example 2 may be used. The Raman detector 1507 may be capable of detecting and identifying single nucleotides 1510 of dATP, dGTP, rhodamine-dCTP and digoxigenin-dUTP moving past the detector 1507. Data on the time course for labeled nucleotide detection may be compiled and analyzed to obtain the sequence of the nucleic acid. In alternative embodiments, the detector 1507 may be capable of detecting and identifying single unlabeled nucleotides.

Example 2 Raman Detection of Nucleotides

Methods and Apparatus

In a non-limiting example, the excitation beam of a Raman detection unit was generated by a titanium: sapphire laser (Mira by Coherent) at a near-infrared wavelength (750˜50 nm) or a gallium aluminum arsenide diode laser (PI-ECL series by Process Instruments) at 785 nm or 830 nm. Pulsed laser beams or continuous beams were used. The excitation beam was transmitted through a dichroic mirror (holographic notch filter by Kaiser Optical or a dichromatic interference filter by Chroma or Omega Optical) into a collinear geometry with the collected beam. The transmitted beam passed through a microscope objective (Nikon LU series), and was focused onto the Raman active substrate, or into the resonant chamber, where target analytes (nucleotides or purine or pyrimidine bases) were located.

The Raman scattered light from the analytes was collected by the same microscope objective, and passed the dichroic mirror to the Raman detector. The Raman detector comprised a focusing lens, a spectrograph, and an array detector. The focusing lens focused the Raman scattered light through the entrance slit of the spectrograph. The spectrograph (Acton Research) comprised a grating that dispersed the light by its wavelength. The dispersed light was imaged onto an array detector (back-illuminated deep-depletion CCD camera by RoperScientific). The array detector was connected to a controller circuit, which was connected to a computer for data transfer and control of the detector function.

For surface-enhanced Raman spectroscopy (SERS), the Raman active substrate consisted of metal-coated nanostructures. Silver nanoparticles, ranging in size from 5 to 200 nm, were made by the method of Lee and Meisel (J. Phys. Chem., 86:3391, 1982). Alternatively, samples were mixed with metallic nanoparticles and were placed on an aluminum substrate under the microscope objective. The following discussion assumes a stationary sample on the aluminum substrate. The number of molecules detected was determined by the optical collection volume of the illuminated sample and the average concentration of the illuminated sample.

Single nucleotides may also be detected by SERS using microfluidic channels. In various embodiments of the invention, nucleotides may be delivered to a Raman active substrate through a microfluidic channel (between about 5 and 200 μm wide). Microfluidic channels can be made by molding polydimethylsiloxane (PDMS), using the technique disclosed in Anderson et al. (“Fabrication of topologically complex three-dimensional microfluidic systems in PDMS by rapid prototyping,” Anal. Chem. 72:3158-3164, 2000).

Where SERS was performed in the presence of silver nanoparticles, the nucleotide, purine or pyrimidine analyte was mixed with LiCl (90 μM final concentration) and nanoparticles (0.25 M final concentration silver atoms). SERS data were collected using room temperature analyte solutions.

Results

Nucleoside monophosphates, purines and pyrimidines were analyzed by SERS, using the system disclosed above. Table 1 shows exemplary detection limits for various analytes of interest.

TABLE 1 SERS Detection of Nucleoside Monophosphates, Purines and Pyrimidines Number of Final Molecules Analyte Concentration Detected dAMP 9 picomolar (pM) ~1 molecule Adenine  9 pM ~1 molecule dGMP  90 μM 6 × 10⁶ Guanine 909 pM 60 dCMP 909 μM 6 × 10⁷ Cyotosine  90 nM 6 × 10³ dTMP  9 μM 6 × 10⁵ Thymine  90 nM 6 × 10³

Conditions were optimized for adenine nucleotides only. LiCL (90 μM final concentration) was determined to provide optimal SERS detection of adenine nucleotides. Detection of other nucleotides may be facilitated by use of other alkali-metal halide salts, such as NaCl, KCl, RbCl or CsCl. The claimed methods are not limited by the electrolyte solution used, and it is contemplated that other types of electrolyte solutions, such as MgCl₂, CaCl₂, NaF, KBr, LiI, etc. may be of use. The skilled artisan will realize that electrolyte solutions that do not exhibit strong Raman signals will provide minimal interference with SERS detection of nucleotides. The results demonstrate that the Raman detection system and methods disclosed above were capable of detecting and identifying single molecules of nucleotides and purine bases. This demonstrates Raman detection of unlabeled nucleotides at the single nucleotide level.

Example 3 Raman Emission Spectra of Nucleotides, Purines and Pyrimidines

The Raman emission spectra of various analytes of interest was obtained using the protocol of Example 2, with the indicated modifications. FIG. 16 shows the Raman emission spectra of a 100 mM solution of each of the four nucleoside monophosphates, in the absence of surface enhancement and without Raman labels. No LiCl was added to the solution. A 10 second data collection time was used. Lower concentrations of nucleotides may be detected with longer collection times, with surface enhancement, using labeled nucleotides and/or with added electrolyte solution. Excitation occurred at 514 nm. For each of the following figures, a 785 nm excitation wavelength was used. As shown in FIG. 16, the unenhanced Raman spectra showed characteristic emission peaks for each of the four unlabeled nucleoside monophosphates.

FIG. 17 shows the SERS spectrum of a 1 nm solution of guanine, in the presence of LiCl and silver nanoparticles. Guanine was obtained from dGMP by acid treatment, as discussed in Nucleic Acid Chemistry, Part 1, L. B. Townsend and R. S. Tipson (eds.), Wiley-Interscience, New York, 1978. The SERS spectrum was obtained using a 100 msec data

FIG. 18 shows the SERS spectrum of a 10 nM cytosine solution, obtained from dCMP by acid hydrolysis. Data were collected using a 1 second collection time.

FIG. 19 shows the SERS spectrum of a 100 nM thymine solution, obtained by acid hydrolysis of dTMP. Data were collected using a 100 msec collection time.

FIG. 20 shows the SERS spectrum of a 100 pM adenine solution, obtained by acid hydrolysis of dAMP. Data were collected for 1 second.

FIG. 21 shows the SERS spectrum of a 500 nM solution of dATP (lower trace) and fluorescein-labeled dATP (upper trace). dATP-fluorescein was purchased from Roche Applied Science (Indianapolis, Ind.). The Figure shows a strong increase in SERS signal due to labeling with fluorescein.

Example 4 Resonance Enhanced Raman Detection of Single Molecules

This example demonstrates that the energies of Raman scattered light generated from single molecules in resonant chambers may be detected by various known radiation detection devices. The results are based on simulations performed by the inventors using MATLAB® available from The MathWorks, Inc. of Natick, Mass.

FIG. 22 shows a plot comparing the energies of Raman scattered light generated from single molecules respectively contained inside an optical resonance chamber (curves A and B), or positioned in free space without resonance enhancement (curve C), according to one embodiment of the invention. The energies, in Joules, are plotted on the y-axis, against time, in seconds, on the x-axis.

The curves A and B plot the energy of Raman scattered light stored in the optical resonance chamber as a function of time. For both curves, the optical resonance chamber is capable of resonating the excitation light as well as the Raman scattered light in order to provide even greater resonant enhancement and stronger signals for detection. The simulations assumed a cavity quality factor of about 10⁶ for both excitation light and Raman scattered light. Even higher cavity quality factors are known in the arts. For example, cavity factors of about 10⁹ have been reported by Spillane et al., in Nature, 415:621-623 (2002). The simulations assumed a Raman cross-section of about 10⁻²⁹ cm². The excitation light was provided as a 1 W continuous-wave. The time steps were 100 nanoseconds.

Different hypothetical molecules with widely differing Raman gain coefficients were used to generate the curves A and B. The Raman gain coefficients essentially quantify the molecules likelihood of generating stimulated Raman emissions. The curve A is based on a molecule with a relatively high Raman gain coefficient of about 20×10⁻⁸ cm/W, which is typical for silicon. The curve B is based on a molecule with zero Raman gain coefficient. As such, the curves A and B respectively provide near upper and lower bounds for the stimulated Raman behavior of most molecules, including nucleic acid derivatives. The energy of the cumulative Raman scattered light generated during the given period of time is plotted in curve C. There is no resonance enhancement in curve C.

As shown, the energies of Raman scattered light generated from the single molecules in the resonant chambers are significantly greater than the energy of the molecule positioned in free space (without resonance enhancement). The greatest energies were observed when the stimulated Raman process was induced (curve A). The energies for the stimulated Raman (curve A) were around eight orders of magnitude larger than the energies without resonance (curve C). Even when the stimulated Raman process was not induced, the energies for the resonated spontaneous Raman process (curve B) were still about six orders of magnitude larger than the energies without resonance (curve C). Such enhancement in the energy available for detection is quite significant.

A dashed line at 10⁻²⁰ J indicates a typical detection level for a variety of commercially available high-quality radiation detection devices. Avalanche photodiodes, photomultiplier tubes, and intensified charge-coupled devices typically have at least such sensitivities. The Raman scattered light generated inside the optical resonance chamber in both curves A and B exceed the indicated detection level within about 100 microseconds. In contrast, the energy of Raman scattered light from the molecule in free space without optical resonance (curve C), remains below the detection level for more than one second.

Accordingly, as demonstrated by these simulations, the energies of Raman scattered light generated from single molecules in resonant chambers may be detected by various known radiation detection devices. Significantly, this is true for both spontaneous Raman (curve B) and stimulated Raman (Curve A).

Example 5 Resonance Enhanced Raman Detection of Multiple Molecules

This prospective example demonstrates how to achieve even greater detection energies than those reported in Example 4 by using a plurality of molecules, instead of just a single molecule. In this approach, a plurality of molecules may be introduced into the resonant chamber. For example, at least 100, or at least 1000 molecules are introduced into the resonant chamber. In one aspect, enough molecules may be introduced to allow detection of a particular type of molecule with a desired radiation detection device. To aid in identification, it may be appropriate to include a majority, or a vast majority, of the molecules being of the same type.

The total detection energy generally increases for each additional molecule in the resonant chamber. Accordingly, each additional molecule should further increase the total energy of Raman scattered light in the resonant chamber. Often, the total energy of Raman scattered light generated in the resonant chamber may initially increase substantially linearly for small numbers of molecules, and then increase even more dramatically (nonlinearly) for larger numbers of molecules. In this way, a plurality of molecules may be used to provide even stronger Raman emission signals.

Thus, spectroscopic analysis chambers and methods for analyzing samples within those chambers are disclosed. While the invention has been described in terms of several embodiments, those skilled in the art will recognize that the invention is not limited to the embodiments described, but may be practiced with modification and alteration within the spirit and scope of the claims. It is to be realized that the optimum dimensional relationships for the parts of the invention, to include variations in size, materials, shape, form, function and manner of operation, assembly and use, are deemed readily apparent to one of ordinary skill in the art, and all equivalent relationships to those illustrated in the drawings and described in the specification are intended to be encompassed by the present invention. The description is thus to be regarded as illustrative instead of limiting. 

What is claimed is:
 1. An apparatus for nucleic acid sequencing comprising: a reaction chamber having an immobilization surface for attachment of a nucleic acid molecule to be sequenced; a microfluidic channel coupled to the reaction chamber for transporting nucleotides released from the nucleic acid molecule; a microchannel coupled to the microfluidic channel at an entrance for receiving the nucleotides, a region of nanoparticles positioned within the microchannel, the region of nanoparticles being configured to allow passage of the nucleotides; an excitation source proximate the region of nanoparticles, the excitation source being configured to excite the nucleotides passing through the region of nanoparticles; and a detector configured to detect the excited nucleotides; wherein the detector comprises an analysis chamber comprising a resonant cavity to contain the nucleotides for analysis, a first window to the cavity to transmit a first electromagnetic radiation having a first frequency into the cavity and to transmit a second electromagnetic radiation having a second frequency out of the cavity, a second window to the cavity to transmit a transverse electromagnetic radiation having the second frequency into the cavity and a plurality of reflectors affixed to a housing of the cavity to reflect radiation of a predetermined frequency, the plurality of reflectors separated by a distance that is sufficient to resonate the radiation of the predetermined frequency.
 2. The apparatus of claim 1, further comprising a pair of electrodes configured to create an electrical potential gradient proximate the entrance to drive the nucleotides from the microfluidic channel into the microchannel.
 3. The apparatus of claim 1, wherein the nanoparticles are cross-linked.
 4. The apparatus of claim 1, wherein the nanoparticles are gold nanoparticles.
 5. The apparatus of claim 1, wherein the nanoparticles are silver nanoparticles.
 6. The apparatus of claim 1, wherein the size of the nanoparticles are from 5 nm to 60 mm.
 7. The apparatus of claim 1, wherein the excitation source is a laser.
 8. The apparatus of claim 1 wherein: the cavity comprises a micro-sized cavity contained within a solid substrate; the micro-sized cavity has a volume that is not greater than one microliter; and the cavity is coupled with a micro-sized fluid channel to receive the nucleotides for analysis.
 9. The apparatus of claim 1 wherein the first window comprises a partial reflector of the plurality of reflectors to transmit the second electromagnetic radiation out of the cavity.
 10. The apparatus of claim 1, wherein the plurality of reflectors comprises a reflector and a partial reflector, the reflector having a reflectivity for the first and second radiations that is greater than 99%, and the partial reflector having a reflectivity for the second radiation that is not greater than 99%.
 11. The apparatus of claim 1, wherein: the plurality of reflectors comprise a multi-layer dielectric mirror; and the second radiation comprises inelastically scattered Raman radiation.
 12. An apparatus for nucleic acid sequencing comprising: a reaction chamber having an immobilization surface for attachment of a nucleic acid molecule to be sequenced; a microfluidic channel coupled to the reaction chamber for transporting nucleotides released from the nucleic acid molecule; a microchannel coupled to the microfluidic channel at an entrance for receiving the nucleotides, a region of nanoparticles positioned within the microchannel, the region of nanoparticles being configured to allow passage of the nucleotides; an excitation source proximate the region of nanoparticles, the excitation source being configured to excite the nucleotides passing through the region of nanoparticles; and a detector configured to detect the excited nucleotides; wherein the detector comprises a spectroscopic analysis chamber comprising: a resonant cavity to contain the nucleotides for analysis, the resonant cavity being configured to receive input excitation radiation in the form of aligned seed radiation and transverse radiation; a first window to the cavity to transmit the seed radiation into the cavity and to transmit an output scattered radiation out of the cavity, a second window to the cavity to transmit a transverse electromagnetic radiation having a second frequency into the cavity, and a plurality of reflectors affixed to a housing of the cavity to reflect radiation of a predetermined frequency, the plurality of reflectors separated by a distance that is sufficient to resonate the radiation of the predetermined frequency.
 13. The apparatus of claim 12, further comprising a pair of electrodes configured to create an electrical potential gradient proximate the entrance to drive the nucleotides from the microfluidic channel into the microchannel.
 14. The apparatus of claim 12, wherein the nanoparticles are cross-linked.
 15. The apparatus of claim 12, wherein the nanoparticles are gold nanoparticles.
 16. The apparatus of claim 12, wherein the nanoparticles are silver nanoparticles.
 17. The apparatus of claim 12, wherein the size of the nanoparticles are from 5 nm to 60 mm.
 18. The apparatus of claim 12, wherein the excitation source is a laser.
 19. The apparatus of claim 12, wherein: the cavity comprises a micro-sized cavity contained within a solid substrate; the micro-sized cavity has a volume that is not greater than one microliter; and the cavity is coupled with a micro-sized fluid channel to receive the nucleotides for analysis.
 20. The apparatus of claim 12, wherein the seed radiation is opposite, but aligned with, the direction of the output scattered radiation.
 21. The apparatus of claim 12, wherein the direction of the transverse radiation into the cavity is perpendicular to the direction of the output scattered radiation out of the cavity.
 22. The apparatus of claim 12, wherein said plurality of reflectors comprises a reflector and a partial reflector and wherein said first window comprises said partial reflector.
 23. The apparatus of claim 22, wherein said reflector and said partial reflector are disposed in a parallel and opposed orientation and are separated by a distance that is sufficient to resonate the output scattered radiation.
 24. The apparatus of claim 12, wherein the plurality of reflectors comprise a multi-layer dielectric mirror. 